The major goal of this proposal is to understand the molecular mechanism(s) of liver fibrosis by hepatitis C virus (HCV) infection. Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV infection often leads to chronic hepatitis in up to 60-80% of infected adults and progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. The mechanisms underlying the liver injury and fibrosis in chronic hepatitis C are unclear. The HCV genome is a positive-sense single stranded RNA molecule that encodes a polyprotein precursor of ~3000 amino acids which is post- translationally cleaved by host and virus-encoded proteases into structural proteins (core, E1, and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). Recently, we have shown the elevated levels of ROS in cultured cells expressing HCV nonstructural proteins. This proposal's main hypothesis is that HCV-induced oxidative stress and Ca2+ signaling induce the synthesis and activation of transforming growth factor-21 (TGF-21) and its potential role in HCV replication and liver fibrosis. To test this hypothesis, the goal is to delineate the signaling pathways in induction of TGF-21 using recently described HCV cell culture infection system. The specific aims are to: 1) To define the kinetics of TGF-21 activation by different HCV genotypes (type 1b vs. 2a). 2) To analyze the role of HCV-induced Ca2+ signaling and oxidative stress in stimulating TGF-21 synthesis. 3) To define the role of cellular kinases and transcription factors on trans-regulation of TGF-21 gene expression. 4) To determine the effect of HCV-induced endogenous bioactive TGF-21 on HCV replication. The stimulation of TGF- 21 expression by HCV-induced oxidative stress and Ca2+ signaling, will be monitored by analyzing the RNA and protein levels of TGF- 21 from HCV infected cells and those treated with antioxidant and Ca2+ chelators. Next, TGF- 21 mediated regulation of HCV replication will be monitored by measuring the HCV RNA levels using quantitative RT- PCR. The assembly of HCV ribonucleoprotein complex (RNP) in response to TGF-21 will be examined by using two-step affinity purification of RNP complexes from HCV expressing cells silenced with TGF-21 siRNA. The proposed studies will yield novel insights into mechanisms of activation of profibrogenic factors that leads to hepatic fibrosis, cirrhosis and pave the way for delineating the pathways preceding liver cancer. PUBLIC HEALTH RELEVANCE: HCV infection often leads to chronic hepatitis which progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. HCV infected hepatocytes display oxidative stress which may provide a link between HCV infection and activation of TGF-21 that leads to progression of fibrosis. The information resulting from these investigations has a potential to provide clues to how HCV-induced oxidative stress and calcium signaling may induce profibrogenic factor, TGF-21, liver fibrosis and ultimately liver cancer.